Trichoderma promoter

ABSTRACT

A promoter for use in producing proteins in filamentous fungal host cells is provided. In one embodiment, the promoter comprises SEQ ID NO:1, or a variant or a truncated form thereof that has promoter activity in a host cell. Also provided are recombinant nucleic acids, vectors containing the promoter and host cells containing a recombinant nucleic acid or vector. Methods of producing a protein using the host cells are also provided.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No.60/971,807 filed Sep. 12, 2007.

FIELD OF THE INVENTION

A promoter for use in producing proteins in filamentous fungal hostcells is provided. In one embodiment, the promoter comprises SEQ IDNO:1, or a variant of SEQ ID NO:1 or a truncated form thereof that haspromoter activity in a host cell. Also provided are recombinant nucleicacids and vectors containing the promoter, and host cells containing arecombinant nucleic acid or vector. Methods of producing a protein usingthe host cells are also provided.

BACKGROUND OF THE INVENTION

Molecular biotechnology is a discipline that is based on the ability ofresearchers to transfer specific units of genetic information from oneorganism to another with the goal of producing commercially relevantamounts of useful products. One of the goals of this cloning process isto achieve maximum expression of the cloned gene. Recombinant productionof a product encoded by a gene is accomplished by constructingexpression vectors suitable for use in a host cell in which the nucleicacid coding for a desired product is placed under the expression controlof a promoter. The expression vector is introduced into a host cell byvarious techniques, such as transformation, and production of thedesired product is then achieved by culturing the transformed host cellunder suitable conditions necessary for the functioning of the promoterincluded in the expression vector.

SUMMARY OF THE INVENTION

A promoter for use in producing proteins in a host cell is provided. Inone embodiment, the promoter comprises SEQ ID NO:1 or a variant ortruncated form thereof that has promoter activity in the host cell. Alsoprovided are recombinant nucleic acids and vectors containing thepromoter, and host cells containing a recombinant nucleic acid orvector. Methods of producing a protein using the host cells are alsoprovided.

In certain cases, the promoter may be employed in filamentous fungalcells to express a protein. In some embodiments, the subject promoter isactive in growth media containing glucose as a sole carbon source. Assuch, in certain cases, the promoter may be active in a growth mediumthat does not contain cellulose, lactose, sophorose, cellobiose, orother sugars or cellulose-related material that are known to induceactivity of cellulase gene expression (see, e.g., Ilmen et al, Appliedand Environmental Microbiology 1997 63: 1298-1306), although suchinducers may be present in addition to glucose. In addition, the subjectpromoter may, in certain cases, be highly active at 37° C., as well aslower temperatures (e.g., 30° C.).

In certain embodiments, the promoter may comprise the nucleotidesequence of: a) SEQ ID NO: 1; b) a subsequence of SEQ ID NO: 1 thatretains promoter activity; or c) a nucleic acid sequence that hybridizesunder stringent hybridization conditions with SEQ ID NO: 1, or asubsequence thereof. In particular embodiments, the nucleotide sequencemay be at least 80% identical (e.g., at least 90%, at least 95%, atleast 98%, or at least 99% identical) to the nucleotide sequence of SEQID NO: 1. In certain cases, the promoter may by identified byhybridizing the promoter of SEQ ID NO:1 with nucleic acid of a differentspecies. In other cases, the promoter may be identified as beingupstream of a nucleic acid that hybridizes to the coding sequence of SEQID NO:2. Hybridization may be done in solution or in silico (by BLAST,etc), for example.

A recombinant nucleic acid comprising the subject promoter is alsoprovided. In certain cases, the recombinant nucleic acid may comprise asubject promoter and a polynucleotide, where the promoter and thepolynucleotide are operably linked such that the promoter causestranscription of the polynucleotide in a cell. In certain cases, thepolynucleotide may contain a coding sequence for a protein. The proteinmay be an enzyme, a reporter or a therapeutic protein (e.g., an antibodyprotein), for example. In certain embodiments, the protein may be afusion protein which may, in certain cases, contain a signal sequence orcarrier portion for secretion of the protein.

A nucleic acid vector comprising the subject recombinant nucleic acid isalso provided, as well as a host cell containing the same. In certainembodiments, the recombinant nucleic acid may be present in the genomeof the host cell or, in other embodiments, the recombinant nucleic acidmay be present in a vector that replicates in the cell. The host cellmay be any of a variety of different host cells, including Trichodermasp, Aspergillus sp., Penicillium sp., Neurospora sp., E. coli, Bacillussp., Streptomyces sp. and Fusarium sp. host cells. In one embodiment,the host cell may be a filamentous fungal host cell.

A culture of cells comprising culture medium and a subject host cell isalso provided.

A method of producing a protein is also provided. In general terms, thismethod includes maintaining a subject culture of cells under conditionssuitable to produce the protein. This method may further includerecovering the protein from culture medium.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a schematic drawing of the plasmid pPGamdS.

FIG. 2 shows two panels of SDS-PAGE gels of culture supernatants from T.reesei strain PGamdS-8 stained with Coomassie Brilliant Blue. M,molecular weight markers; lane 1, growth on lactose as carbon source;lane 2, growth on glucose/sophorose as carbon source; lane 3, growth onglucose as carbon source. A, cultures grown at 28° C.; B, cultures grownat 37° C.

FIG. 3 shows the nucleotide sequences of SEQ ID NOS:1, 2 and 3, and theamino acid sequence of SEQ ID NO:4. The three underlined nucleotides inSEQ ID NO:1 are not present in the stp1 promoter amplified from T.reesei. The sequences shown in bold are potential transcription factorbinding sites.

FIG. 4 is a schematic drawing of the plasmid pKB429.

DETAILED DESCRIPTION Definitions

Unless defined otherwise herein, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention belongs. Singleton, et al.,DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY, 2D ED., John Wiley andSons, New York (1994), and Hale & Marham, THE HARPER COLLINS DICTIONARYOF BIOLOGY, Harper Perennial, N.Y. (1991) provide one of skill withgeneral dictionaries of many of the terms used in this invention.Although any methods and materials similar or equivalent to thosedescribed herein can be used in the practice or testing of the presentinvention, the preferred methods and materials are described.

All patents and publications, including all sequences disclosed withinsuch patents and publications, referred to herein are expresslyincorporated by reference.

Numeric ranges are inclusive of the numbers defining the range. Unlessotherwise indicated, nucleic acids are written left to right in 5′ to 3′orientation; amino acid sequences are written left to right in amino tocarboxy orientation, respectively.

The headings provided herein are not limitations of the various aspectsor embodiments of the invention which can be had by reference to thespecification as a whole. Accordingly, the terms defined immediatelybelow are more fully defined by reference to the specification as awhole.

The term “promoter” is defined herein as a nucleic acid that directstranscription of a downstream polynucleotide in a cell. In certaincases, the polynucleotide may contain a coding sequence and the promotermay direct the transcription of the coding sequence into translatableRNA.

The term “promoter activity” is defined herein as the ability of anucleic acid to direct transcription of a downstream polynucleotide in ahost cell. To test promoter activity, the nucleic acid may be operablylinked to a polynucleotide to produce a recombinant nucleic acid. Therecombinant nucleic acid may be transferred into a cell andtranscription of the polynucleotide may be evaluated. In certain cases,the polynucleotide may encode a protein, and transcription of thepolynucleotide can be evaluated by assessing production of the proteinin the cell. As will be discussed in greater detail below, the host cellmay be a filamentous fungal host cell, e.g., a T. reesei host cell.

The term “functional equivalent”, with reference to a promoter, isdefined herein as a promoter having a nucleic acid sequence comprising asubstitution, deletion and/or insertion in one or more nucleotides of aparent promoter. The term “functionally equivalent promoter” includesnaturally-occurring equivalents and in vitro generated equivalents. Afunctionally equivalent promoter need not have a promoter activity thatis identical to a parent promoter. The functionally equivalent promotermay have more promoter activity, less promoter activity or the samepromoter activity compared to the corresponding parent promoter. As usedherein the term “variant” promoter is used interchangeability withfunctional equivalent promoter.

The term “hybrid promoter” as defined herein means parts of two or morepromoters which are fused together resulting in a sequence which is afusion of two or more promoters and having promoter activity whichresults in the transcription of a downstream polynucleotide.

The term “tandem promoter” is defined herein as two or more promoterseach of which is operably linked to a coding sequence of interest.

The term “isolated” as defined herein means a compound, a protein, cell,nucleic acid sequence or amino acid that is removed from at least onecomponent with which it is naturally associated.

The term “coding sequence” is defined herein as a nucleic acid that,when placed under the control of appropriate control sequences includinga promoter, is transcribed into mRNA which can be translated into apolypeptide. A coding sequence may contain a single open reading frame,or several open reading frames separated by introns, for example. Acoding sequence may be cDNA, genomic DNA, synthetic DNA or recombinantDNA, for example. A coding sequence generally starts at a start codon(e.g., ATG) and ends at a stop codon (e.g., UAA, UAG and UGA).

The term “recombinant” refers to a polynucleotide or polypeptide thatdoes not naturally occur in a host cell. A recombinant molecule maycontain two or more naturally occurring sequences that are linkedtogether in a way that does not occur naturally.

The term “heterologous” refers to elements that are not normallyassociated with each other. For example, a heterologous protein is aprotein that is not produced in a wild-type host cell, a heterologouspromoter is a promoter that is not present in nucleic acid that isendogenous to a wild type host cell, and a promoter operably linked to aheterologous coding sequence is a promoter that is operably linked to acoding sequence that it is not usually operably linked to in a wild-typehost cell.

The term “operably linked” refers to a juxtaposition, wherein elementsare in an arrangement allowing them to be functionally related. Forexample, a promoter is operably linked to a coding sequence if itcontrols the transcription of the sequence, and a signal sequence isoperably linked to a protein if the signal sequence directs the proteinthrough the secretion system of a host cell.

The term “nucleic acid” encompasses DNA, RNA, single or doubled strandedand modification thereof. The terms “nucleic acid” and “polynucleotide”may be used interchangeability herein.

The term “DNA construct” as used herein means a nucleic acid sequencethat comprises at least two DNA polynucleotide fragments.

As used herein, the term “reporter” refers to a protein that is easilydetected and measured. In certain cases, a reporter may be opticallydetectable, e.g., fluorescent, luminescent or colorigenic.

The term “signal sequence” or “signal peptide” refers to a sequence ofamino acids at the N-terminal portion of a protein, which facilitatesthe secretion of the mature form of the protein outside the cell. Themature form of the extracellular protein lacks the signal sequence whichis cleaved off during the secretion process.

The term “vector” is defined herein as a polynucleotide designed tocarry nucleic acid sequences to be introduced into one or more celltypes. Vectors include cloning vectors, expression vectors, shuttlevectors, plasmids, phage or virus particles, DNA constructs, cassettesand the like. Expression vectors may include regulatory sequences suchas promoters, signal sequences, a coding sequences and transcriptionterminators.

An “expression vector” as used herein means a DNA construct comprising acoding sequence that is operably linked to suitable control sequencescapable of effecting expression of a protein in a suitable host. Suchcontrol sequences may include a promoter to effect transcription, anoptional operator sequence to control transcription, a sequence encodingsuitable ribosome binding sites on the mRNA, enhancers and sequenceswhich control termination of transcription and translation.

As used herein, the terms “polypeptide” and “protein” are usedinterchangeably and include reference to a polymer of any number ofamino acid residues. The terms apply to amino acid polymers in which oneor more amino acid residue is an artificial chemical analog of acorresponding naturally occurring amino acid, as well as to naturallyoccurring amino acid polymers. The terms also apply to polymerscontaining conservative amino acid substitutions such that thepolypeptide remains functional. “Peptides” are polypeptides having lessthan 50 amino acid residues.

A “host cell” is a cell that contains a subject recombinant nucleicacid, either in the genome of the host cell or in an extrachromosomalvector that replicates autonomously from the genome of the host cell. Ahost cell may be any cell type.

The term “filamentous fungi” refers to all filamentous forms of thesubdivision Eumycotina (See, Alexopoulos, C. J. (1962), INTRODUCTORYMYCOLOGY, Wiley, New York). These fungi are characterized by avegetative mycelium with a cell wall composed of chitin, glucans, andother complex polysaccharides. The filamentous fungi of the presentinvention are morphologically, physiologically, and genetically distinctfrom yeasts. Vegetative growth by filamentous fungi is by hyphalelongation and carbon catabolism is obligatory aerobic.

A “non-pathogenic” filamentous fungi is a strain that is not pathogenicto humans.

“Transformation” means introducing DNA into a cell so that the DNA ismaintained in the cell either as an extrachromosomal element orchromosomal integrant.

Promoters

In certain embodiments, a subject promoter comprises the nucleotidesequence of SEQ ID NO: 1, or a subsequence (sometimes referred herein asa truncated promoter) of SEQ ID NO:1 that retains promoter activity. Thesubsequence may contain at least about 100 nucleotides, at least about200 nucleotides; at least about 250 nucleotides; at least about 300nucleotides; at least about 400 nucleotides; at least about 450nucleotides; at least about 450 nucleotides, at least about 500nucleotides, at least about 550 nucleotides, at least about 600nucleotides, at least about 650 nucleotides that are contiguous in SEQID NO:1, including the entire contiguous sequence of SEQ ID NO: 1, or avariant thereof that retains promoter activity. In one embodiment, thefirst about 1 kb of SEQ ID NO: 1 is removed and the promoter stillretains activity, including 1.05 kb, 1.1 kb, 1.2 kb, 1.3 kb, and 1.4 kb.In some embodiments, the truncated promoter includes at least the partof the promoter containing the putative transcription factor bindingsites (see in bold in SEQ ID NO:1). In another embodiment, the truncatedpromoter contains at least the region from the start of the positiveregulatory transcription factor binding sites through the transcriptionstart site.

In certain embodiments, a functional equivalent promoter may include oneor more changes (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, more than 10, up to20, 30, 40 or 50 or more changes) relative to the nucleotide sequence ofSEQ ID NO: 1, where a change can be a deletion, substitution orinsertion, for example. In one exemplary embodiment, the nucleotidesequence of the subject promoter may include one to five or one totwenty nucleotide differences relative to the nucleotide sequence of theSEQ ID NO:1. In one embodiment, the third transcription factor bindingsite of SEQ ID NO:1 (ctgggg) is mutated to remove any inhibitoryactivity. In further embodiments, the first and second transcriptionfactor binding sites are conserved as potential positive regulatoryregions.

In other embodiments, the promoter may include a nucleotide sequencethat hybridizes under stringent hybridization conditions to apolynucleotide having the nucleotide sequence of SEQ ID NO: 1, wherestringent hybridization conditions encompass low, medium, high and veryhigh stringency hybridization conditions. In other embodiments, thepromoter may include a nucleic acid sequence that is upstream from acoding sequence that hybridizes to the coding sequence of SEQ ID NO:2 orSEQ ID NO:3. In these embodiments, the coding sequence that hybridizesto the coding sequence of SEQ ID NO:2 or SEQ ID NO:3 can encode aprotein that is a sugar transporter.

“Low-stringency” conditions refer to washing with a solution of1×SSC/0.1% SDS at 20° C. for 15 minutes. “Medium-stringency” conditionsrefer to washing with a solution of 1×SSC/0.1% SDS at 65° C. for 60minutes. “High-stringency” conditions refer to washing with a solutionof 0.2×SSC/0.1% SDS at 65° C. for 10 minutes. “Very high-stringency”conditions refer to washing with a solution of 0.2×SSC/0.1% SDS at 65°C. for 60 minutes.

Hybridization methods are described in great detail in Sambrook et al.,MOLECULAR CLONING: A LABORATORY MANUAL (2^(nd) Ed., 1989 Cold SpringHarbor, N.Y.). In one exemplary hybridization assay, a DNA sample iselectrophoresed through an agarose gel (for example, 0.8% agarose) sothat of the DNA fragment can be visualized by ethidium bromide staining.The gel is then briefly rinsed in distilled H₂O and subsequentlydepurinated in an appropriate solution (such as, for example, 0.25M HCl)with gentle shaking followed by denaturation for 30 minutes (m, forexample, 0.4 M NaOH) with gentle shaking. A renaturation step may beincluded, in which the gel is placed in 1.5 M NaCl, 1MTris, pH 7.0 withgentle shaking for 30 minutes. The DNA is then transferred onto anappropriate positively charged membrane, for example, Maximum StrengthNytran Plus membrane (Schleicher & Schuell, Keene, N.H.), using atransfer solution (such as, for example, 6×SSC, i.e., 900 mM NaCl, 90 mMtrisodium citrate). Once the transfer is complete, generally after about2 hours, the membrane is rinsed in e.g., 2×SSC (300 mM NaCl, 30 mMtrisodium citrate) and air dried at room temperature. The membrane maybe prehybridized (for approximately 2 hours or more) in a suitableprehybridization solution (such as, for example, an aqueous solutioncontaining per 100 mL: 20-50 mL formamide, 25 mL of 20×SSPE (1×SSPE=0.18M NaCl, 1 mM EDTA, 10 mM NaH₂PO₄, pH 7.7), 2.5 mL of 20% SDS, and 1 mLof 10 mg/mL sheared herring sperm DNA). As would be known to one ofskill in the art, the amount of formamide in the prehybridizationsolution may be varied depending on the nature of the reaction obtainedaccording to routine methods. Thus, a lower amount of formamide mayresult in more complete hybridization in terms of identifyinghybridizing molecules than the same procedure using a larger amount offormamide. On the other hand, a strong hybridization band may be moreeasily visually identified by using more formamide.

A DNA probe generally between 50 and 500 bases in length having at least100 or 200 or more contiguous nucleotides of the nucleic acid of FIG. 1may be isolated by electrophoresis in an agarose gel, the fragmentexcised from the gel, and recovered from the excised agarose. Thispurified fragment of DNA may be labeled (using, for example, theMegaprime labeling system according to the instructions of themanufacturer) to incorporate p³² in the DNA. The labeled probe isdenatured by heating to 95° C. for 5 minutes and immediately added tothe membrane and prehybridization solution. The hybridization reactionshould proceed for an appropriate time and under appropriate conditions,for example, for 18 hours at 37° C. with gentle shaking or rotating. Themembrane is rinsed (for example, in 2×SSC/0.3% SDS) and then washed inan appropriate wash solution, as described above, with gentle agitation.Hybridization can be detected by autoradiography.

In another embodiment, a subject promoter may contain a contiguousnucleotide sequence that is at least 80%, at least 85%, at least 90%, atleast 95%, at least 96%, at least 97%, at least 98% or at least 99%identical to SEQ ID NO: 1, or a subsequence thereof. In one embodiment,the subject promoter may contain a contiguous nucleotide sequence thatis at least 95% identical to SEQ ID NO:1. In a further embodiment, thepromoter may have 80% sequence identity to SEQ ID NO:1 (including 85%,90%, 95%, 97% and 99%) and 100% identity in transcription factor bindingsites 1 and 2.

The term “identity” in the context of two nucleic acid sequences refersto nucleotides residues in the two sequences that are the same whenaligned for maximum correspondence, as measured using any of thefollowing sequence comparison algorithms. Optimal alignment of sequencesfor comparison can be conducted, e.g., by the local homology algorithmof Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homologyalignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970),by the search for similarity method of Pearson & Lipman, Proc. Nat'lAcad. Sci. USA 85:2444 (1988), by computerized implementations of thesealgorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin GeneticsSoftware Package, Genetics Computer Group, 575 Science Dr., Madison,Wis.), or by visual inspection.

An example of an algorithm that is suitable for determining sequencesimilarity is the BLAST algorithm, which is described in Altschul, etal., J. Mol. Biol. 215:403-410 (1990). Software for performing BLASTanalyses is publicly available through the National Center forBiotechnology Information available on the world wide web (www)ncbi.nlm.nih.gov. The BLAST algorithm performs a statistical analysis ofthe similarity between two sequences (see, e.g., Karlin & Altschul,Proc. Nat'l. Acad. Sci. USA 90:5873-5787 (1993)).

As noted in the Examples section below, the nucleic acid of SEQ ID NO: 1was obtained from Trichoderma reesei, a filamentous fungus. As would bereadily apparent, variants of SEQ ID NO: 1 that retain promoter activitycan be identified by identifying sequences that are similar to SEQ IDNO: 1 in other filamentous fungi. Since most or all of the genomesequences of other filamentous fungi, e.g., Aspergillus (e.g.,Aspergillus fumigatus, Aspergillus oryzae (see, e.g., Machida et al,Nature 2005 438, 1157-1161), Aspergillus nidulans, Aspergillusfumigatus, Aspergillus niger, Aspergillus flavus, and Aspergillusterreus), Neurospora (e.g., Neurospora crassa), and Fusarium (e.g.,Fusarium graminearum) are available, functional equivalents of SEQ IDNO: 1 that have promoter activity may be readily identifiable. Suchpromoters should be linked to a polynucleotide encoding a sugartransporter, e.g., a protein having at least 80% identity to SEQ IDNO:4, including 85%, 90%, 95%, 97%, 99% and 100% identity.

As noted above, a subject promoter may have promoter activity in a hostcell. Promoter activity may be detected using any suitable assay. Incertain embodiments, a subject promoter may be operably linked to apolynucleotide, and transcription of the polynucleotide may be detectingusing any suitable method, e.g., Northern blotting or RT-PCR, etc. Inother embodiments, the promoter may be operably linked to apolynucleotide that encodes a protein, e.g., a reporter protein, and theactivity of the promoter can be evaluated by detecting the protein. Inthese embodiments, if necessary, a 5′ untranslated region may be linkedto the promoter such that the resultant transcript has a 5′ UTR followedby a coding sequence. As would be recognized, the results obtained fromsuch an assay may be compared to results compared to a suitable control,e.g., a negative or positive control, to determine the significance ofresults obtained. Any host cell, e.g., a bacterial host cell such as E.coli, Bacillus or Streptomyces host cell, or a filamentous fungal cell,e.g., an Aspergillus ssp., Trichoderma ssp. or Fusarium ssp. host cellmay be employed. There is no requirement for a subject promoter to becontained within a particular host cell. In certain cases, the promotermay be tested for promoter activity in a Trichoderma reesei host cell.

The activity of a subject promoter is generally detectable using theassay employed. In certain cases, the activity of a variant promoter(e.g., a functionally equivalent promoter) may have at least about 20%,at least about 30%, at least about 40%, at least about 50%, at leastabout 60%, at least about 80%, at least about 90%, at least about 95% orat least about 100% of the promoter activity of the promoter of SEQ IDNO: 1 in the same type of cell, e.g., a Trichoderma cell. In some cases,the activity of a variant promoter (e.g., functionally equivalentpromoter) may be greater, for example more than about 100%, more thanabout 150%, more than about 200%, more than about 250%, or more than1000% of the activity of SEQ ID NO: 1 in the same type of cell. In otherembodiments, the promoter has at least about 40%, 50%, 60%, 70%, 80% 90%95%, 97%, and 99% of the activity on a particular carbon source.

In certain embodiments, the promoter may be a hybrid promoter comprisinga portion of a subject promoter and a portion of another promoter. Insome embodiments, the hybrid promoter will include a subsequence of SEQID NO: 1 having at least about 100 nucleotides, at least about 150nucleotides; at least about 200 nucleotide; at least about 250nucleotides; at least about 300 nucleotides, at least about 350nucleotides, at least about 400 nucleotides, at least about 500nucleotides, at least about 550 nucleotides, at least about 600nucleotides, at least about 650 nucleotides, at least about 700nucleotides, at least about 750 nucleotides, at least about 800nucleotides, at least about 850 nucleotides, at least about 900nucleotides, at least about 950 nucleotides, at least about 1000nucleotides, at least about 1050 nucleotides, at least about 1100nucleotides, at least about 1150 nucleotides, at least about 1200nucleotides, at least about 1250 nucleotides, at least about 1300nucleotides, at least about 1350 nucleotides, at least about 1400nucleotides, at least about 1450 nucleotides, at least about 1500nucleotides, at least about 1550 nucleotides, at least about 1600nucleotides, at least about 1650 nucleotides, at least about 1700nucleotides, at least about 1750 nucleotides, at least about 1800nucleotides, at least about 1850 nucleotides, and at least about 1900nucleotides of SEQ ID NO: 1. In certain embodiments, the hybrid promoterwill include a subsequence of SEQ ID NO: 1 comprising the first andsecond transcription factor binding sites.

The other promoter of the hybrid promoter may be any promoter that showspromoter activity in a host cell, and includes mutant promoters,truncated promoters and the like which may or may not be native to thehost cell. Examples of other promoters, which may be useful in a hybridpromoter of the invention, include fungal and bacterial promoters. Somespecific nonlimiting examples include; the aprE promoter or a mutantaprE promoter (WO 01/51643); the aph promoter of the Streptomycesfradiae aminoglycoside 3′-phosphotransferase gene; an Aspergillus nigerglucoamylase (glaA) promoter; the glucose isomerase (GI) promoter ofActinoplanes missouriensis and the derivative GI (GIT) promoter (U.S.Pat. No. 6,562,612 and EPA 351029); the glucose isomerase (GI) promoterfrom Streptomyces lividans, the short wild-type GI promoter, the 1.5 GIpromoter, the 1.20 GI promoter, or any of the variant GI promoters asdisclosed in WO 03/089621; the cbh1, cbh2, egl1 and egl2 promoters fromfilamentous fungi and specifically the Trichoderma reeseicellobiohydrolase I promoter (GenBank Accession No. D86235); theAspergillus niger or A. awamori glucoamylase (glaA) promoter (Nunberg etal. (1984) supra, and Boel et al., (1984) supra); the lacZ and tacpromoters (Bagdasarion et al., 1983, Gene 26:273-282); the ermE promoter(Ward et al., 1986, Mol. Gen. Genet. 203:468-478 and Schmitt-John etal., 1992, Appl. Microbiol. Biotechnol. 36:493-498); and the Bacillussubtilis phage ø29 promoters (Pulido et al., 1986, Gene 49:377-382).Promoters effective in Streptomyces are listed in Hopwood et al.,(Hopwood et al., Regulation of Gene Expression in Antibiotic-producingStreptomyces. In Booth, I. and Higgins, C. (Eds) SYMPOSIUM OF THESOCIETY FOR GENERAL MICROBIOLOGY, REGULATION OF GENE EXPRESSION,Cambridge University Press, 1986 pgs. 251-276). Streptomyces phagepromoters are also disclosed in Labes et al., 1997, Microbiol.143:1503-1512. Other promoters which may be effective for use in thehybrid promoters herein are promoters listed in Deuschle et al., 1986EMBO J. 5:2987-2994 and WO 96/00787.

The promoter may also be a tandem promoter, which comprises two or morepromoters. In some embodiments, the tandem promoter will include thesubject promoter and one or more other promoters such as those discussedabove for hybrid promoters.

Recombinant Nucleic Acids

A subject recombinant nucleic acid may comprise a subject promoter and apolynucleotide encoding a protein (i.e., a coding sequence), where thepromoter and the polynucleotide are operably linked such that theisolated nucleic acid causes transcription of the polynucleotide, and,in certain embodiments, production of the protein.

The encoded protein may be an enzyme, a therapeutic protein, a reporterprotein, a selectable marker, a food additive or a foodstuff or thelike.

Enzyme usage in industrial applications covers a wide array of enzymefunctionalities, for example industrial enzymes include oxidoreductases(e.g., glucose oxidases, catalases, and laccases), transferases (e.g.transglutaminases), hydrolases (e.g., lipases, phytases, amylases,cellulases, xylanases, mannanases, proteases, subtilisins, andaspergillopepsins), lyases (e.g. pectate lyases) and isomerases (e.g.xylose isomerases).

In one embodiment, the protein may be an enzyme such as a carbohydrase,such as a liquefying and saccharifying α-amylase, an alkaline α-amylase,a β-amylase, a cellulase; a dextranase, an α-glucosidase, anα-galactosidase, a glucoamylase, a hemicellulase, a pentosanase, axylanase, an invertase, a lactase, a naringanase, a pectinase or apullulanase; a protease such as an acid protease, an alkali protease,bromelain, ficin, a neutral protease, papain, pepsin, a peptidase,rennet, rennin, chymosin, subtilisin, thermolysin, an asparticproteinase, or trypsin; a lipase or esterase, such as a triglyceridase,a phospholipase, acyl transferase, a pregastric esterase, a phosphatase,a phytase, an amidase, an iminoacylase, a glutaminase, a lysozyme, or apenicillin acylase; an isomerase such as glucose isomerase; anoxidoreductases, e.g., an amino acid oxidase, a catalase, achloroperoxidase, a glucose oxidase, a hydroxysteroid dehydrogenase or aperoxidase; a lyase such as a acetolactate decarboxylase, an asparticβ-decarboxylase, a fumarese or a histadase; a transferase such ascyclodextrin glycosyltransferase; or a ligase, for example.

In particular embodiments, the protein may be an aminopeptidase, acarboxypeptidase, a chitinase, a cutinase, a deoxyribonuclease, anα-galactosidase, a β-galactosidase, a β-glucosidase, a laccase, amannosidase, a mutanase, a pectinolytic enzyme, a polyphenoloxidase,ribonuclease or transglutaminase, for example.

In other particular embodiments, the enzyme will be a α-amylase, acellulase; an α-glucosidase, an α-galactosidase, a glucoamylase, ahemicellulase, a xylanase, a pectinase, a pullulanase; an acid protease,an alkali protease, an aspartic proteinase, a lipase, a cutinase or aphytase.

In another embodiment, the protein may be a therapeutic protein (i.e., aprotein having a therapeutic biological activity). Examples of suitabletherapeutic proteins include: erythropoietin, cytokines such asinterferon-α, interferon-β, interferon-γ, interferon-o, andgranulocyte-CSF, GM-CSF, coagulation factors such as factor VIII, factorIX, and human protein C, antithrombin III, thrombin, soluble IgEreceptor α-chain, IgG, IgG fragments, IgG fusions, IgM, IgA,interleukins, urokinase, chymase, and urea trypsin inhibitor,IGF-binding protein, epidermal growth factor, growth hormone-releasingfactor, annexin V fusion protein, angiostatin, vascular endothelialgrowth factor-2, myeloid progenitor inhibitory factor-1,osteoprotegerin, α-1-antitrypsin, α-feto proteins, DNase II, kringle 3of human plasminogen, glucocerebrosidase, TNF binding protein 1,follicle stimulating hormone, cytotoxic T lymphocyte associated antigen4-Ig, transmembrane activator and calcium modulator and cyclophilinligand, soluble TNF receptor Fc fusion, glucagon like protein 1 and IL-2receptor agonist. Antibody proteins, e.g., monoclonal antibodies thatmay be humanized, are of particular interest.

In a further embodiment, the protein may be a reporter protein. Suchreporter proteins may be optically detectable or colorigenic, forexample. In this embodiment, the protein may be a β-galactosidase(lacZ), β-glucuronidase (GUS), luciferase, alkaline phosphatase,nopaline synthase (NOS), chloramphenicol acetyltransferase (CAT),horseradish peroxidase (HRP) or a fluorescent protein green, e.g., greenfluorescent protein (GFP), or a derivative thereof.

Examples of selectable markers include but are not limited to ones thatconfer antimicrobial resistance (e.g. resistance to hygromycin,bleomycin, chloroamphenicol or phleomycin), and proteins that confermetabolic advantage, e.g., amdS, argB and pyr4. Selectable markers arefurther described in Kelley et al., (1985) EMBO J. 4: 475-479; Penttilaet al., (1987) Gene 61:155-164 and Kinghorn et al (1992) AppliedMolecular Genetics of Filamentous Fungi, Blackie Academic andProfessional, Chapman and Hall, London.

In certain embodiments, the coding sequence may encode a fusion protein.In some of these embodiments, the fusion protein may provide forsecretion of the protein from the host cell in which it is expressedand, as such, may contain a signal sequence operably linked to theN-terminus of the protein, where the signal sequence contains a sequenceof amino acids that directs the protein to the secretory system of thehost cell, resulting in secretion of the protein from the host cell intothe medium in which the host cell is growing. The signal sequence iscleaved from the fusion protein prior to secretion of the protein. Thesignal sequence employed may be endogenous or non-endogenous to the hostcell and, in certain embodiments, may be signal sequence of a proteinthat is known to be highly secreted from a host cell. In particularembodiments, the signal sequence protein may be any signal sequence thatfacilitates protein secretion from a filamentous fungal (e.g.,Trichoderma or Aspergillus) host cell. Such signal sequences include,but are not limited to: the signal sequence of cellobiohydrolase I,cellobiohydrolase II, endoglucanase I, endoglucanase II, endoglucanaseIII, α-amylase, aspartyl proteases, glucoamylase, mannanase, glycosidaseand barley endopeptidase B (see Saarelainen, Appl. Environ. Microbiol.1997 63: 4938-4940), for example. Other of signal sequences are thoseoriginating from the α factor gene (yeasts e.g. Saccharomyces,Kluyveromyces and Hansenula) or the α amylase gene (Bacillus). Incertain embodiments, therefore, the subject recombinant nucleic acid maycomprise: a signal sequence-encoding nucleic acid operably linked to aprotein-encoding nucleic acid, where translation of the nucleic acid ina host cell produces a fusion protein comprising a protein having anN-terminal signal sequence for secretion of the protein from the hostcell.

In particular embodiments, the fusion protein may further contain a“carrier protein”, which is a portion of a protein that is endogenous toand highly secreted by the host cell. Suitable carrier proteins includethose of T. reesei mannanase I (Man5A, or MANI), T. reeseicellobiohydrolase II (Cel6A, or CBHII) (see, e.g., Paloheimo et al Appl.Environ. Microbiol. 2003 December; 69(12): 7073-7082) or T. reeseicellobiohydrolase I (CBHI). In one embodiment, the carrier protein is atruncated T. reesei CBH1 protein that includes the CBH1 core region andpart of the CBH1 linker region. A fusion protein containing, fromamino-terminus to carboxy-terminus, a signal sequence, a carrier proteinand a subject protein in operable linkage is therefore provided, as wellas a nucleic acid encoding the same.

In certain embodiments, the polynucleotide may be codon optimized forexpression of the protein in a particular host cell. Since codon usagetables listing the usage of each codon in many cells are known in theart (see, e.g., Nakamura et al, Nucl. Acids Res. 2000 28: 292) orreadily derivable, such nucleic acids can be readily designed giving theamino acid sequence of a protein to be expressed.

In addition to a coding sequence, the recombinant nucleic acid may incertain embodiments further contain other elements that are necessaryfor expression of the protein in the host cell. For example, the nucleicacid may contain a transcriptional terminator, and 5′ and 3′ UTRsequences. Suitable 5′ UTR sequences may be obtained from the T. reeseicbh1, cbh2, egl1, egl2, egl5, xln1 and xln2 genes, for example. Suitableterminators include the T. reesei cbh1, cbh2, egl1, egl2, egl5, xln1 andxln2 terminators, and many others, including, for example, theterminators from A. niger or A. awamori glucoamylase genes (Nunberg etal. (1984) supra, and Boel et al., (1984) supra), Aspergillus nidulansanthranilate synthase genes, Aspergillus oryzae TAKA amylase genes, orA. nidulans trpc (Punt et al., (1987) Gene 56:117-124). The promoterand/or terminator may be native or non-endogenous to the host cell. Incertain cases, the promoter and protein coding sequence may be separatedby a sequence encoding a 5′ untranslated region, for example.

As will be discussed in greater detail below, a subject recombinantnucleic acid may be present in a vector, or integrated into a genome(i.e., the nuclear genome) of a host cell.

Vectors

A subject recombinant nucleic acid may be present in a vector, e.g., aphage, plasmid, viral, or retroviral vector that autonomously replicatesin a host cell. In certain embodiments, the vector may be an expressionvector for expressing a protein in a host cell. In certain embodiments,the vector may be an expression vector for expressing a subjectpolypeptide in a filamentous fungal cell.

Vectors for expression of recombinant proteins are well known in the art(Ausubel, et al, Short Protocols in Molecular Biology, 3rd ed., Wiley &Sons, 1995; Sambrook, et al., Molecular Cloning: A Laboratory Manual,Second Edition, (1989) Cold Spring Harbor, N.Y.).

A subject vector may be constructed using well known techniques as isgenerally described for example in EPO publication 0 215 594. Once thefusion DNA construct is made it may be incorporated into any number ofvectors as is known in the art. While the DNA construct will preferablyinclude a promoter sequence, in some embodiments the vector will includeregulatory sequences functional in the host to be transformed, such aspromoters, ribosomal binding sites, transcription start and stopsequences, terminator sequences, polyadenylation signals, enhancers andor activators.

Terminator sequences which are recognized by the expression host toterminate transcription may be operably linked to the 3′ end of thefusion DNA construct encoding the fusion protein to be expressed. Thoseof general skill in the art are well aware of various terminatorsequences that may be used with filamentous fungi. Non-limiting examplesinclude the terminator from the Aspergillus nidulans trpC gene (YeltonM. et al., (1984) Proc. Natl. Acad. Sci. USA 81: 1470-1474) theterminator from the Aspergillus niger glucoamylase genes (Nunberg et al.(1984) Mol. Cell. Biol. 4: 2306-2353).

In further embodiments, the fusion DNA construct or the vectorcomprising the fusion DNA construct will contain a selectable markergene to allow the selection of transformed host cells. Selection markergenes are well known in the art and will vary with the host cell used.Examples of selectable markers include but are not limited to ones thatconfer antimicrobial resistance (e.g. hygromycin, bleomycin,chloroamphenicol and phleomycin). Sequences that confer metabolicadvantage, such as nutritional selective markers also find use. Also,sequences encoding proteins that complement an auxotrophic defect may beused as selection markers (e.g. pyr4 complementation of a pyr4 deficientA. nidulans, A. awamori or Trichoderma reesei and argB complementationof an argB deficient strain). Reference is made to Kelley et al., (1985)EMBO J. 4: 475-479; Penttila et al., (1987) Gene 61:155-164 and Kinghomet al (1992) Applied Molecular Genetics of Filamentous Fungi, BlackieAcademic and Professional, Chapman and Hall, London.

In one embodiment, the vector is a Trichoderma expression vector relatedto pTrex3g, which is described in detail in Example 6 of WO 05/001036.

Host Cells

A host cell comprising a subject recombinant nucleic acid is alsoprovided. The host cell may be any cell type, e.g., bacterial (such asE. coli, Bacillus sp. or Streptomyces sp.) or fungal (such as anon-filamentous or filamentous fungal) host cell. In certainembodiments, the host cell may be a filamentous fungal host cell. Insome embodiments, the host cell may be a cell of a strain that has ahistory of use for production of proteins that has GRAS status, i.e., aGenerally Recognized as Safe, by the FDA.

In particular embodiments, the subject host cell may be a fungal cell ofthe following species: Trichoderma, (e.g., Trichoderma reesei(previously classified as T. longibrachiatum and currently also known asHypocrea jecorina), Trichoderma viride, Trichoderma koningii, andTrichoderma harzianum)); Penicillium sp., Humicola sp. (e.g., Humicolainsolens and Humicola grisea); Chrysosporium sp. (e.g., C. lucknowense),Gliocladium sp., Aspergillus sp. (e.g., Aspergillus oryzae, Aspergillusniger, Aspergillus nidulans, Aspergillus kawachi, Aspergillus aculeatus,Aspergillus japonicus, Aspergillus sojae, and Aspergillus awamori),Fusarium sp., Humicola sp, Mucor sp., Neurospora sp., Hypocrea sp., orEmericella sp. (See also, Innis et al., (1985) Sci. 228:21-26), amongothers. Other host cells include Bacillus sp., including, but notlimited to B. subtilis, B. licheniformis, B. lentus, B. brevis, B.stearothermophilus, B. alkalophilus, B. amyloliquefaciens, B. clausii,B. halodurans, B. megaterium, B. coagulans, B. circulans, B. lautus, andB. thuringiensis, and Steptomyces sp., including, but not limited to: S.lividans, S. carbophilus and S. helvaticus.

In some embodiments, subject fungal host cells may be of a strain ofAspergillus niger which include ATCC 22342, ATCC 44733, ATCC 14331, ATCC11490, NRRL 3112, and strains derived therefrom. In some embodiments,subject fungal cells may be strains of Trichoderma which includefunctional equivalents of RL-P37 (Sheir-Neiss et al. (1984) Appl.Microbiol. Biotechnology 20:46-53). Other useful host strains include;NRRL 15709, ATCC 13631, ATCC 26921 (QM 9414) ATCC 32098, ATCC 32086, andATCC 56765 (RUT-30).

In some embodiments, a host cell may be one wherein native genes havebeen deleted or inactivated. For example genes corresponding to proteasegenes (e.g. aspartyl protease) (Berka et al. (1990) Gene 86:153-162 andU.S. Pat. No. 6,509,171 or genes corresponding to cellulase genes may bedeleted or inactivated, (e.g. cbh1, cbh2 and egl1, and egl2) such as thequad deleted strain of T. reesei disclosed in WO 05/001036.

The above described fusion DNA construct may be present in the nucleargenome of the host cell or may be present in a plasmid that replicatesin the host cell, for example.

Introduction of a nucleic acid into a host cell includes techniques suchas transformation; electroporation; nuclear microinjection;transduction; transfection, (e.g., lipofection mediated and DEAE-Dextrinmediated transfection); incubation with calcium phosphate DNAprecipitate; high velocity bombardment with DNA-coated microprojectiles;and protoplast fusion. General transformation techniques are known inthe art (See, e.g., Ausubel et al., (1987), supra, chapter 9; andSambrook (1989) supra, and Campbell et al., (1989) Curr. Genet.16:53-56). Reference is also made to WO 05/001036; U.S. Pat. No.6,022,725; U.S. Pat. No. 6,103,490; U.S. Pat. No. 6,268,328; andpublished U.S. patent applications 20060041113, 20060040353, 20060040353and 20050208623, which publications are incorporated herein byreference.

The expression of recombinantly introduced proteins in Trichoderma isdescribed in U.S. Pat. No. 6,022,725; U.S. Pat. No. 6,268,328; Harkki etal. (1991); Enzyme Microb. Technol. 13:227-233; Harkki et al., (1989)Bio Technol. 7:596-603; EP 244,234; EP 215,594; and Nevalainen et al.,“The Molecular Biology of Trichoderma and its Application to theExpression of Both Homologous and Heterologous Genes”, in MOLECULARINDUSTRIAL MYCOLOGY, Eds. Leong and Berka, Marcel Dekker Inc., NY (1992)pp. 129-148). Reference is also made to Cao et al., (2000) Protein Sci.9:991-1001; Yelton et al., (1984) Proc. Natl. Acad. Sci. 81:1470-1471;U.S. Pat. No. 6,590,078; and Berka, et al., (1991) in: Applications ofEnzyme Biotechnology, Eds. Kelly and Baldwin, Plenum Press, NY) fortransformation of Aspergillus strains.

In one embodiment, the preparation of Trichoderma sp. for transformationinvolves the preparation of protoplasts from fungal mycelia. (See,Campbell et al., (1989) Curr. Genet. 16:53-56). In some embodiments, themycelia are obtained from germinated vegetative spores. Transformationand protein expression in Aspergillus and Trichoderma is furtherdescribed in, for example U.S. Pat. No. 5,364,770; U.S. Pat. No.6,022,725; and Nevalainen et al., 1992, The Molecular Biology ofTrichoderma and its Application to the Expression of Both Homologous andHeterologous Genes, in MOLECULAR INDUSTRIAL MYCOLOGY, Eds. Leon andBerka, Marcel Dekker, Inc. pp. 129-148.

A culture of cells is also provided. The culture of cells may contain apopulation of the above-described cells, and growth medium. The growthmedium may contain glucose as a carbon source. In particularembodiments, glucose may be the sole carbon source of the growth medium.The growth medium may be free of a carbon source that is known to induceactivity of cellulase gene expression (see, e.g., Ilmen et al, Appliedand Environmental Microbiology 1997 63: 1298-1306). For example, thegrowth medium may be free of cellulose, lactose, sophorose, cellobiose,and/or other sugar or cellulose-related material that induce cellulaseexpression. The culture of cells may be at a temperature of about 30° C.(e.g., 27-33° C.), or at a temperature of about 37° C. (e.g., 34-39°C.), for example. In a particular embodiment, the growth medium maycontain glucose, glucose and sopohorose, or lactose as a carbon source,and the culture may be grown at 30° C. or 37° C.

Protein Production

Methods of using the above-described cells are also provided. Theproteins produced by the cells may be employed in a variety of methods.

In certain embodiments, the subject methods include: culturing the cellsto produce a recombinant protein. In certain embodiments and asdiscussed above, the protein may be secreted into the culture medium. Assuch, certain embodiments of the method include the step of recoveringthe protein from the culture medium.

Cells may cultured in a standard medium containing physiological saltsand nutrients (See, e.g., Pourquie, J. et al., BIOCHEMISTRY AND GENETICSOF CCELLULOSE DEGRADATION, eds. Aubert, J. P. et al., Academic Press,pp. 71-86, 1988 and Ilmen, M. et al., (1997) Appl. Environ. Microbiol.63:1298-1306). Common commercially prepared media (e.g., Yeast MaltExtract (YM) broth, Luria Bertani (LB) broth and Sabouraud Dextrose (SD)broth also find use in the present invention. Preferred cultureconditions for a given filamentous fungus are known in the art and maybe found in the scientific literature and/or from the source of thefungi such as the American Type Culture Collection (ATCC) and FungalGenetics Stock Center.

In some embodiments, a subject host cell may be cultured under batch orcontinuous fermentation conditions. A classical batch fermentation is aclosed system, wherein the composition of the medium is set at thebeginning of the fermentation and is not subject to artificialalterations during the fermentation. Thus, at the beginning of thefermentation the medium is inoculated with the desired organism(s). Inthis method, fermentation is permitted to occur without the addition ofany components to the system. Typically, a batch fermentation qualifiesas a “batch” with respect to the addition of the carbon source andattempts are often made at controlling factors such as pH and oxygenconcentration. The metabolite and biomass compositions of the batchsystem change constantly up to the time the fermentation is stopped.Within batch cultures, cells progress through a static lag phase to ahigh growth log phase and finally to a stationary phase where growthrate is diminished or halted. If untreated, cells in the stationaryphase eventually die. In general, cells in log phase are responsible forthe bulk of production of end product.

A variation on the standard batch system is the “fed-batch fermentation”system, which also finds use with the present invention. In thisvariation of a typical batch system, the substrate is added inincrements as the fermentation progresses. Fed-batch systems are usefulwhen catabolite repression is apt to inhibit the metabolism of the cellsand where it is desirable to have limited amounts of substrate in themedium. Measurement of the actual substrate concentration in fed-batchsystems is difficult and is therefore estimated on the basis of thechanges of measurable factors such as pH, dissolved oxygen and thepartial pressure of waste gases such as CO₂. Batch and fed-batchfermentations are common and known in the art.

Continuous fermentation is an open system where a defined fermentationmedium is added continuously to a bioreactor and an equal amount ofconditioned medium is removed simultaneously for processing. Continuousfermentation generally maintains the cultures at a constant high densitywhere cells are primarily in log phase growth.

Continuous fermentation allows for the modulation of one factor or anynumber of factors that affect cell growth and/or end productconcentration. For example, in one embodiment, a limiting nutrient suchas the carbon source or nitrogen source is maintained at a fixed rateand all other parameters are allowed to moderate. In other systems, anumber of factors affecting growth can be altered continuously while thecell concentration, measured by media turbidity, is kept constant.Continuous systems strive to maintain steady state growth conditions.Thus, cell loss due to medium being drawn off must be balanced againstthe cell growth rate in the fermentation. Methods of modulatingnutrients and growth factors for continuous fermentation processes aswell as techniques for maximizing the rate of product formation areknown.

A fungal host cell may be cultured in a standard medium containingphysiological salts and nutrients (See, e.g., Pourquie, J. et al.,BIOCHEMISTRY AND GENETICS OF CELLULOSE DEGRADATION, eds. Aubert, J. P.et al., Academic Press, pp. 71-86, 1988 and Ilmen, M. et al., (1997)Appl. Environ. Microbiol. 63:1298-1306). Common commercially preparedmedia (e.g., Yeast Malt Extract (YM) broth, Luria Bertani (LB) broth andSabouraud Dextrose (SD) broth also find use in the present methods.Preferred culture conditions for fungal host cells are known in the artand may be found in the scientific literature and/or from the source ofthe fungi such as the American Type Culture Collection (ATCC) and FungalGenetics Stock Center.

Protein may be recovered from growth media by any convenient method,e.g., by precipitation, centrifugation, affinity, filtration or anyother method known in the art. In another embodiment, a culture of cellsis provided, where the culture of cells comprises: a) growth medium andb) the above-described host cell.

As noted above, the cells may be grown using glucose as a carbon sourcewhich, in certain embodiments, may be the sole carbon source for thecells. The growth medium may be free of cellulose, lactose, sophorose,cellobiose, and/or other sugar or cellulose-related material that inducecellulase expression. The cells may be cultured at a temperature ofabout 30° C. (e.g., 27-33° C.), or at a temperature of about 37° C.(e.g., 34-39° C.), for example.

In order to further illustrate the present invention and advantagesthereof, the following specific examples are given with theunderstanding that they are being offered to illustrate the presentinvention and should not be construed in any way as limiting its scope.

Example 1 Identification of the Trichoderma reesei stp1 Gene

The stp1 gene was identified from the Trichoderma reesei genome sequencedata made publicly available by the United States Department of EnergyJoint Genome Institute (JGI) and accessible at the websitegenome.jgi-psf.org/Trire2/Trire2.home. Several gene models thatannotated the gene with the translation start and stop codons andintrons were proposed by JGI. Manual inspection of these models suggeststhat the model labeled estExt_fgenesh1_pg.C_(—)30027, associated withthe Protein ID 43977, is the most likely to be correct. All of theelements (transcription factor binding sites, transcription start site,etc.) of the promoter for this gene are expected to reside withinapproximately 2 kb sequence that is immediately upstream, or 5′, of thetranslation initiation codon. The sequence of this 2 kb promoter regionas extracted from the JGI genome data is shown as SEQ ID NO: 1. However,it is likely that the promoter will still be active with the removal ofat least the first 1 kb (or more) of SEQ ID NO: 1. Further, 3 possibletranscription factor binding sites (regulatory sites) were identifiedand are shown in FIG. 3 in SEQ ID NO:1 in bold. The site closest to thetranscription start site (Site 3) is a potential repressor bindingregion. The other sites (Site 1 and 2) are positive regulatory sites.

The sequence of the open reading frame of the stp1 gene, includingintrons, is shown as SEQ ID NO:2 and the open reading frame with threepredicted introns removed is shown as SEQ ID NO:3. The deduced aminoacid sequence of the encoded STP1 protein is shown as SEQ ID NO:4.

The STP1 protein sequence has similarity with sugar transporter proteinsof the major facilitator superfamily. Twelve transmembrane regions arepredicted by topology prediction algorithms such as TMHMM (website:cbs.dtu.dk/services/TMHMM-2.0/) and Prosite motifs corresponding tosugar transport proteins can be recognized within the amino acidsequence (website expasy.org/prosite/).

Expression of the stp1 gene in different T. reesei strains and duringgrowth under a variety of conditions was investigated by examining datafrom transcript profiling experiments using microarrays (e.g., Foremanet al., 2003, J. Biol. Chem. 278:31988-31997). These data suggested thatthe stp1 gene is highly expressed under conditions that are known toinduce cellulase and hemicellulase production in Trichoderma reesei suchas growth in the presence of cellulose or lactose or as a result ofinduction by sophorose. Expression of stp1 is low when abundant glucoseis present as the carbon source. However, unlike cellulase geneexpression, stp1 expression is increases dramatically at the point whenglucose is exhausted from the medium. These features of stp1 generegulation made the stp1 promoter attractive for directing expression ofgenes encoding desired proteins in Trichoderma reesei.

Example 2 Construction of a Vector for Expression of the Trichodermareesei Glucoamylase Gene Using the Stp1 Promoter

A vector, pPGamdS, was designed for the expression of an open readingframe encoding the T. reesei glucoamylase. The promoter region from thestp1 gene was amplified by PCR using the following primer pair.

Primer newpF: (SEQ ID NO: 5) 5′-ggccaagcttgagctgagtgtcaaggcagttgcacPrimer newpR: (SEQ ID NO: 6) 5′-gggaccgcggtaatctctagcctctgggccagagac

These primers were designed to amplify the stp1 promoter and introduce aHindIII restriction site at the 5′ and a SacII cleavage site at the 3′end seven nucleotides upstream from the translation start codon. Thetemplate for the PCR reaction was genomic DNA isolated from Trichodermareesei. Pfu Turbo DNA polymerase (Stratagene Corp.) was used accordingto the manufacturer's instructions. The following temperatures and timeswere used for the thermocycling steps of the PCR. 95° C. for 30 seconds;followed by 30 cycles of 95° C. for 30 seconds, 55° C. for 30 secondsand 68° C. for 2 minutes; and a final step of 68° C. for 7 minutes. Anapproximately 2 kb DNA fragment was obtained and was cloned into plasmidpCR-BluntII-TOPO (InVitrogen Life Technologies) according to thesupplier's instructions. DNA sequence analysis confirmed that the stp1promoter had been cloned. Except for the absence of three base pairs inthe DNA sequence of the cloned stp1 promoter, the sequence was identicalto that in the JGI Trichoderma reesei database. The nucleotide positionsare 1278, 1279 and 1280, relative to SEQ ID NO:1.

The cloned stp1 promoter was next fused to an open reading frameencoding the Trichoderma reesei glucoamylase using a PCR fusionstrategy. The DNA sequence and the polypeptide sequence of theTrichoderma reesei glucoamylase is disclosed in WO 06/060062 publishedJun. 6, 2006 and reference is made to SEQ ID NO:1, SEQ ID NO:2 and SEQID NO: 4 therein.

The cloned stp1 promoter was first amplified from pCR-BluntII-TOPO usingthe following primer pair.

Primer newpF and Primer PGfuse1-r: (SEQ ID NO: 7)5′-gtcgacaggacgtgcattgttaccgcggtaatctctagcctctg

The Trichoderma reesei gla1 open reading frame (approximately 2 kb inlength), encoding glucoamylase, was amplified using the following primerpair.

Primer PGfuse1: (SEQ ID NO: 8)5′-cagaggctagagattaccgcggtaacaatgcacgtcctgtcgac Primer trgaR:(SEQ ID NO: 9) 5′-cgcggcgcgccttacgactgccaggtgtcctccttg

The products from the above two amplification reactions were mixed andserved as template in a subsequent reaction using the following primerpair.

-   -   Primer newpF and Primer trgaR

The approximately 4 kb product from this amplification reaction was afragment of DNA consisting of the stp1 promoter linked to the gla1coding region and having a HindIII restriction site at the 5′ end and anAscI restriction site at the 3′ end. This 4 kb DNA fragment was clonedinto pCR-BluntII-TOPO and was subsequently excised as a HindIII-AscIfragment for insertion into a Trichoderma expression vector to createpPGamdS (FIG. 1).

In pPGamdS the T. reesei glucoamylase open reading frame is flanked bythe stp1 promoter and the T. reesei cbh1 gene terminator sequences. Thevector is based on the bacterial plasmid pNEB193 (New England Biolabs)and also contains the Aspergillus nidulans amdS gene, encodingacetamidase, with its native promoter and terminator sequences for useas a selectable marker for transformation of T. reesei.

Plasmid pPGamdS was inserted into a Trichoderma reesei strain derivedfrom RL-P37 (Sheir-Neiss, G. and Montenecourt, B. S., 1984, Appl.Microbiol. Biotechnol. 20:46-53) and deleted for the cbh1, cbh2, egl1,and egl2 genes as described by Bower et al (Carbohydrases fromTrichoderma reesei and other micro-organisms, Royal Society ofChemistry, Cambridge, 1998, p. 327-334). The plasmid was inserted intospores of T. reesei using a biolistic transformation procedure.DNA-coated tungsten particles were prepared as follows. 60 mg of M10tungsten particles were added to 1 ml ethanol (70% or 100%) in amicrocentrifuge tube. This mixture was allowed to soak for 15 minutes,followed by centrifugation for 15 min at 15,000 rpm. The supernatant wasthen decanted and the pellet washed three times with sterile distilledwater. The majority of the distilled water was removed after the finalwash. The pellet was then resuspended in 1 ml of a 50% glycerol (v/v,sterile) solution. While continuously vortexing a 25 ul aliquot of thisparticle suspension was removed and placed in a microcentrifuge tube. Tothis tube the following components were added (while continuouslyvortexing) in the following order. 0.5-5 ul of pPGamdS DNA solution (1ug/ul), 25 ul 2.5M CaCl₂, and 10 ul 0.1M spermidine. The mixture wasallowed to coat the particles for 5-15 minutes during continuousvortexing, and was used as soon as possible to avoid tungstendegradation of the DNA. The mixture was then centrifuged forapproximately three seconds. The supernatant was then removed and thepellet was washed with approx 200 ul of 70% ethanol (v/v) followed by a3 second centrifugation and removal of the supernatant. The pellet wasagain washed with 200 ul of 100% ethanol, followed by another 3 secondcentrifugation. The supernatant was removed and the pellet was thenresuspended in 24 ul 100% ethanol and mixed by pipetting. 8 ul aliquotswere placed onto macrocarrier discs (Bio-Rad, Hercules, Calif.) bypipetting the aliquots in the exact center of the disks while the diskswere in a dessicator. The discs were kept in a dessicator untilthoroughly dry and kept there until immediately before use. Themacrocarrier discs were inserted into a Model PDS-1000/He BiolisticParticle Delivery System (Bio-Rad, Hercules, Calif.). This apparatus wasused according to the manufacturer's directions to propel the DNA-coatedtungsten particles at the T. reesei spores prepared as below.

A spore suspension of strain the Trichoderma strain was made withapproximately 5×10⁸ spores/ml. 100-200 ul aliquots of the sporesuspension was spread over an area approximately 6 cm in diameter at thecenter of a plate of agar medium containing acetamide as sole nitrogensource. After the biolistic transformation, the plates were placed in a28° C. incubator for 4 days. Transformant colonies were able to grow dueto incorporation and expression of the amdS gene encoding acetamidase.Transformants were transferred onto fresh agar plates with acetamide assole nitrogen source and incubated at 28° C. before transfer to liquiddefined culture medium.

Liquid defined (LD) culture medium contained the following components.Casamino acids, 9 g/L; (NH₄)₂SO₄, 5 g/L; MgSO₄.7H₂O, 1 g/L; KH₂PO₄, 4.5g/L; CaCl₂.2H₂O, 1 g/L, PIPPS, 33 g/L, 400×T. reesei trace elements, 2.5ml/L; pH adjusted to 5.5 with NaOH. 400×T. reesei trace elementssolution contained the following: citric Acid (anhydrous), 175 g/L;FeSO₄.7H₂O, 200 g/L, ZnSO₄.7H₂O, 16 g/L, CuSO₄.5H₂O, 3.2 g/L; MnSO₄.H₂O,1.4 g/L; H₃BO₃, 0.8 g/L. After sterilization, lactose, glucose or aglucose/sophorose mixture was added to a final concentration of 1.6%w/v.

Twenty four morphologically stable transformant colonies on agar mediumwere inoculated into LD medium with lactose. After 5 days of growth at28° C. the secreted proteins were analyzed by polyacrylamide gelelectrophoresis (SDS-PAGE) of culture supernatant samples. Thosetransformants that showed an obvious band on SDS-PAGE corresponding insize to the T. reesei glucoamylase protein, which was absent in culturesupernatant from the T. reesei parent strain, were identified.Transformant PGamdS-8 was chosen as the best producer of glucoamylase.

Transformant PGamdS-8 was cultured in shake flasks under a variety ofconditions to determine the effect of carbon source and temperature onglucoamylase production directed by the stp1 promoter. From a colony onagar medium one square cm was excised and used to inoculate 50 ml LDmedium with glucose in a baffled 250 ml shake flask. After 2 days ofgrowth at 28° C. and 200 rpm, 5 ml of this pre-culture was used toinoculate shake flasks of 50 ml LD medium with lactose,glucose/sophorose mixture or glucose as carbon source. This productionculture was grown for 4 days at 28° C. or 37° C. and 200 rpm.Supernatants were collected by centrifugation of the fermentation brothand glucoamylase production was assessed by SDS-PAGE.

As shown in FIG. 2 a high level of production of glucoamylase wasobserved when lactose or a mixture of glucose plus sophorose was used ascarbon source. Glucoamylase was also observed when glucose was the solecarbon source, albeit at a reduced level, and production was apparentwhen cultures were grown at either 28° C. or 37° C.

Example 3 Expression of Cerrena unicolor Laccase in Trichoderma reeseiUsing the stp1 Promoter

Expression of the laccase D gene from Cerrena unicolor in Trichodermareesei was disclosed in United States provisional application GC993P,Ser. No. 60/984,430, “Improved heterologous protein production in a hostusing signal sequences and co-expressing chaperones” by Genencor, ADanisco Division (herein incorporated by reference in its entirety),which also describes plasmid pKB410. Laccase D expression is furtherdescribed in WO 08/076,322 published Jun. 28, 2008.

Plasmid pKB410 contains the T. reesei cbh1 promoter functionally fusedto an open reading frame encoding the T. reesei CBHI signal sequencefused to the mature laccase D protein. The plasmid also contains theAspergillus nidulans amdS gene for selection of transformants in T.reesei. The cbh1 promoter was removed from pKB410 by digestion withHindIII and SacII and replaced with the 2 kb HindIII-SacII fragment frompPGamdS bearing the T. reesei stp1 promoter to create pKB429 (FIG. 4).Plasmids pKB410 and pKB429 were inserted independently into theTrichoderma reesei strain by the biolistic transformation procedure asdescribed in Example 2. Ten stable transformants with pKB410 and 14stable transformants with pKB429 were isolated and screened for secretedlaccase D production by measuring activity on ABTS as described inUnited States provisional application GC993P, “Improved heterologousprotein production in a host using signal sequences and co-expressingchaperones” by Genencor, A Danisco Division. The four highest producingtransformants with each plasmid (designated as clones PCBH1-1, PCBH1-3,PCBH1-6, and PCBH1-9 with pKB410 and Pstp1-2, Pstp1-3, Pstp1-4, andPstp1-11 with pKB429) were chosen for further study. These transformantswere cultured in shake flasks in 50 ml LD medium with lactose as carbonsource at 28° C. Supernatant samples were taken each day on days 2through 8 and the laccase activity on ABTS was measured. As can be seenfrom Table 1, laccase production using the stp1 promoter was higher thanthat using the cbh1 promoter.

TABLE 1 Comparison of Laccase D production (ABTS activity insupernatant) cbh1 promoter stp1 promoter Clone Day PCBH1-1 PCBH1-3PCBH1-6 PCBH1-9 Pstp1-2 Pstp1-3 Pstp1-4 Pstp1-11 2 0.03 0.02 0.04 0.020.04 0.05 0.03 0.07 3 0.23 0.19 0.23 0.17 0.27 0.27 0.31 0.32 4 0.770.50 0.81 0.58 1.01 1.18 0.60 1.59 5 1.55 2.37 1.59 1.27 2.16 2.42 3.682.79 6 2.11 1.84 2.23 1.72 3.10 3.18 5.98 3.68 7 2.74 2.27 2.51 2.083.58 3.84 7.00 4.48 8 3.09 2.51 2.85 2.25 3.92 4.18 8.45 5.12

All publications and patents mentioned in the above specification areherein incorporated by reference. Various modifications and variationsof the described methods and system of the invention will be apparent tothose skilled in the art without departing from the scope and spirit ofthe invention. Although the invention has been described in connectionwith specific preferred embodiments, it should be understood that theinvention as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications of the described modes forcarrying out the invention which are obvious to those skilled in the artare intended to be within the scope of the following claims.

1. An isolated nucleic acid comprising the nucleotide sequence of SEQ IDNO: 1 or a variant or a truncated form thereof that has promoteractivity in a filamentous fungal host cell wherein the variant comprisesa sequence that is at least 90% identical to SEQ ID NO:1.
 2. A promotercomprising a nucleotide sequence that is at least 95% identical to thenucleotide sequence of SEQ ID NO:
 1. 3. The isolated nucleic acid ofclaim 1, wherein said nucleic acid hybridizes under high stringencyconditions with a polynucleotide having the nucleotide sequence of SEQID NO:
 1. 4. A recombinant nucleic acid comprising the isolated nucleicacid of claim 1, claim 2, or claim 3 and a polynucleotide encoding aprotein, wherein said isolated nucleic acid and said polynucleotide areoperably linked such that said isolated nucleic acid causestranscription of said polynucleotide in filamentous fungal host cell. 5.The recombinant nucleic acid of claim 4, wherein said protein is anenzyme.
 6. The recombinant nucleic acid of claim 5, wherein said enzymeis a glucoamylase, an amylase, a cellulase, a protease, a xylanase, alipase, a phytase, a hemicellulase, a pectinase, a catalase, an oxidase,a glucanase, a glycosidase, or a laccase.
 7. The recombinant nucleicacid of claim 4, wherein said protein is a therapeutic protein.
 8. Anucleic acid vector comprising the recombinant nucleic acid of claim 4.9. A host cell comprising the recombinant nucleic acid of claim
 4. 10.The host cell of claim 9, wherein said host cell is an Aspergillus sp.,a Trichoderma sp., a Humicola sp., or a Fusarium sp. host cell.
 11. Thehost cell of claim 10, wherein said host cell is a Trichoderma sp. hostcell.
 12. The host cell of claim 11, wherein the Trichoderma sp. is aTrichoderma reesei.
 13. A method of producing a protein comprising,transforming a filamentous fungal host cell with a recombinant nucleicacid of claim 4, culturing the host cell under suitable cultureconditions to allow the expression and production of the protein. 14.The method of claim 13 further comprising recovering said protein fromsaid culture.